The goal of this SBIR project is to develop a new drug discovery platform for screening tyrosine kinase inhibitors. The main innovation of the proposed platform is the use of a multi-enzyme assay, which is able to measure efficiency and identify inhibition mechanism in a single experiment in real-time using a small-volume sample. The proposed platform addresses the drawbacks of the existing methods by reducing the cost of reagents by ten times or more;by substantially reducing the assay time and significantly increasing the information yield of the drug screening process. The technology is adaptable to high through put format and has unique capabilities for identification and study allosteric kinase inhibitors, which play a central role in the development of new pharmaceuticals for treatment of cancer, inflammatory diseases, neural disorders, and metabolism problems. PUBLIC HEALTH RELEVANCE: Protein kinases represent one of the most promising groups of drug targets due to the vital role played by kinases in such pathological conditions as cancer, inflammatory diseases, neural disorders, and metabolism problems. Currently about 25% of all research spending on drug discovery and development is directed to new kinase inhibitors with a special focus on the discovery of allosteric inhibitors, which have a higher inhibition efficiency, lower toxicity and longer duration of action. Yet, identification of new allosteric inhibitors is challenging and requires tedious kinetic and X-ray diffraction studies, which are costly, labor intensive and too often are not carried out at the full extend. The technology under development in this SBIR project has a unique capability for identification and study allosteric kinase inhibitors and is able to provide more information quickly and at a lower cost.